Crosslinkable macromers bearing initiator groups

ABSTRACT

A crosslinkable macromer system that includes two or more polymer-pendent polymerizable groups and one or more polymer-pendent initiator groups. The polymerizable groups and the initiator group(s) can be pendent on the same or different polymeric backbones. The macromer system provides advantages over the use of polymerizable macromers and separate, low molecular weight initiators, including advantages with respect to such properties as nontoxicity, efficiency, and solubility.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of prior application Ser. No.60/078,607 filed Mar. 19, 1998.

TECHNICAL FIELD

The present invention relates to the preparation of matrices by thepolymerization of macromers. In another aspect, the invention relates tothe use of such matrices for such purposes as cell immobilization,tissue adherence, and controlled drug delivery.

BACKGROUND OF THE INVENTION

Matrices are polymeric networks characterized by insolubility in water.One type of polymeric matrix is a hydrogel, which can be defined as awater-containing polymeric network. The polymers used to preparehydrogels can be based on a variety of monomer types, such as thosebased on methacrylic and acrylic ester monomers, acrylamide(methacrylamide) monomers, and N-vinyl-2-pyrrolidone. To form the gel,these monomer classes are typically crosslinked with such crosslinkingagents as ethylene dimethacrylate, N,N'-methylenediacrylamide,methylenebis(4-phenyl isocyanate), ethylene dimethacrylate,divinylbenzene, and allyl methacrylate.

Another type of polymeric network can be formed from more hydrophobicmonomers and/or macromers. Matrices formed from these materialsgenerally exclude water. Polymers used to prepare hydrophobic matricescan be based on a variety of monomer types such as alkyl acrylates andmethacrylates, and polyester-forming monomers such as ε-caprolactone andlactide. When formulated for use in an aqueous environment, thesematerials do not need to be crosslinked, but they can be crosslinkedwith standard agents such as divinyl benzene. Hydrophobic matrices canalso be formed from reactions of macromers bearing the appropriatereactive groups such as the reaction of diisocyanate macromers withdihydroxy macromers, and the reaction of diepoxy-containing macromerswith dianhydride or diamine-containing macromers.

Although there exist a variety of methods for producing polymericnetworks, when these networks are intended to be created in the presenceof viable tissue, and/or to contain a bioactive compound, the number ofacceptable methods of producing polymeric networks is extremely limited.

It is nevertheless desirable to form both hydrogel and non-hydrogelpolymeric matrices in the presence of viable tissue or bioactive agentsfor the purposes of drug delivery, cellular immune isolation, preventionof post-surgical adhesions, tissue repair, and the like. These polymericmatrices can be divided into two categories: biodegradable orbioresorbable polymer networks and biostable polymer networks.

Biodegradable polymeric matrices have been previously suggested for avariety of purposes, including controlled release carriers, adhesivesand sealers. When used as controlled release carriers, for instance,polymeric matrices can contain and release drugs or other therapeuticagents over time. Such matrices can be formed, for instance, by a numberof different processes, including solvent casting hydrophobic polymers.Solvent casting, however, typically involves the use of organic solventsand/or high temperatures which can be detrimental to the activity ofbiological materials and can complicate production methods. Solventcasting of polymers out of solution also results in the formation ofuncrosslinked matrices. Such matrices have less structure thancrosslinked matrices and it is more difficult to control the release ofbioactive agents from such matrices. Yet another process, which involvesthe polymerization of monomers in or around the desired materials,suffers from cytotoxicity of monomers, oxygen inhibition and heat ofpolymerization complications.

Another process used in the past to prepare biodegradable and biostablehydrogels involves the polymerization of preformed macromers using lowmolecular weight initiators. This process involves a number of drawbacksas well, however, including toxicity, efficacy, and solubilityconsiderations. For instance, when using a macromer solution containinga low molecular weight soluble initiator to encapsulate viable cellularmaterial, the initiator can penetrate the cellular membrane and diffuseinto the cells. The presence of the initiator may involve some toxicconsequence to the cells. When activated, however, these initiatorsproduce free radicals having distinct cytotoxic potential. Otherdrawbacks arise if the initiator is able to diffuse out of the formedmatrix, thereby producing toxicity and other issues. Such initiatorsalso tend to aggregate in aqueous solution, causing efficiency andreproducibility problems. Finally, in view of the limited efficiency ofmany initiators for initiating the necessary radical chainpolymerization, it is often necessary to add one or more monomericpolymerization "accelerators" to the polymerization mixture. Suchaccelerators tend to be small molecules capable of penetrating thecellular membrane, and often raise cytotoxic or carcinogenic concerns.

U.S. Pat. Nos. 5,410,016 (Hubbell, et al.) and 5,529,914 (Hubbell,et.al.) for instance, relate to hydrogels prepared from biodegradableand biostable polymerizable macromers. The hydrogels are prepared fromthese polymerizable macromers by the use of soluble, low molecularweight initiators. Such initiators can be combined with the macromers,and irradiated in the presence of cells, in order to form a gel thatencapsulates the cells.

Hydrogels often suffer from similar or other drawbacks in use asbiological adhesives or sealants, e.g., for use as tissue adhesives,endovascular paving, prevention of post-surgical adhesions, etc. In eachof the applications, the hydrogel matrix must generally "adhere" to oneor more tissue surfaces. Current methods rely upon physical "adhesion"or the tendency of hydrogels to "stick" to a surface. A superioradhesive would provide both physical and chemical adhesion to surfacesutilizing the same physical characteristics as current hydrogeladhesives, but also providing chemical, covalent coupling of the matrixmaterial to the tissue surface. Covalent bonds are generally muchstronger than physical adhesive forces, such as hydrogen bonding and vander Waals forces.

As described above, when various techniques are used to form polymericmatrices via photoinitiation of macromers, the photoinitiators utilizedtend to be low molecular weight. Polymeric photoinitiators have beendescribed as well, although for applications and systems quite distinctfrom those described above. See, for instance, "Radical Polymerization",C. H. Bamford, pp. 940-957 in Kroschwitz, ed., Concise Encyclopedia ofPolymer Science and Engineering, 1990. In the subsection entitled"Photosensitized Initiation: Polymeric Photosensitizers andPhotoinitiators", the author states that "[p]olymeric photosensitizersand photoinitiators have been described. Many of these are polymersbased on benzophenone, e.g., poly(p-divinylbenzophenone) (DVBP). Suchrigid polymers are reported to be effective sensitizers since hydrogenabstraction from the backbone by excited benzophenone is less likely."

U.S. Pat. No. 4,315,998 (Neckers) describes polymer-boundphotosensitizing catalysts for use in the heterogeneous catalysis ofphotosensitized chemical reactions such as photo-oxidation,photodimerization, and photocyclo addition reactions. The polymer-boundphotosensitizing catalysts are insoluble in water and common organicsolvents, and therefore can be readily separated from the reactionmedium and reaction products by simple filtration.

What is clearly needed are macromers and macromer systems that avoid theproblems associated with conventional polymeric matrices, and inparticular, those drawbacks that arise when polymeric matrices areformed in the presence of viable tissue or bioactive agents.

SUMMARY OF THE INVENTION

The present invention provides a crosslinkable macromer systemcomprising two or more polymer-pendent polymerizable groups and one ormore polymer-pendent initiator groups. In a preferred embodiment, thepolymerizable groups and the initiator group(s) are pendent on the samepolymeric backbone. In an alternative preferred embodiment, thepolymerizable groups and initiator group(s) are pendent on differentpolymeric backbones.

In the first embodiment, the macromer system comprises a polymericbackbone to which are covalently bonded both the polymerizable groupsand initiator group(s). Pendent initiator groups can be provided bybonding the groups to the backbone at any suitable time, e.g., eitherprior to the formation of the macromer (for instance, to monomers usedto prepare the macromer), or to the fully formed macromer itself. Themacromer system itself will typically comprise but a small percentage ofmacromers bearing both initiator groups and polymerizable groups. Themajority of macromers will provide only pendent polymerizable groups,since the initiator groups are typically sufficient if present at farless than 1:1 stoichiometric ratio with macromer molecules.

In an alternative preferred embodiment, the macromer system comprisesboth polymerizable macromers, generally without pendent initiatorgroups, in combination with a polymeric initiator. In either embodiment,the initiator will be referred to herein as a "polymeric initiator", byvirtue of the attachment of such initiator groups to a polymericbackbone.

Macromer systems of the present invention, employing polymericinitiators, provide a number of unexpected advantages over the use ofpolymerizable macromers and separate, low molecular weight initiators.Such systems, for instance, provide an optimal combination of suchproperties as nontoxicity, efficiency, and solubility. Solubility, forinstance, can be improved by virtue of the ability to control theaqueous or organic solubility of the polymerizable macromer bycontrolling the backbone. Toxicity can also be improved, since thepolymeric initiators of this invention typically cannot diffuse intocells in the course of immobilization.

In a preferred embodiment, the pendent initiator groups are selectedfrom the group consisting of long-wave ultra violet (LWUV)light-activatable molecules such as; 4-benzoylbenzoic acid,[(9-oxo-2-thioxanthanyl)-oxy]acetic acid, 2-hydroxy thioxanthone, andvinyloxymethylbenzoin methyl ether; visible light activatable molecules;eosin Y, rose bengal, camphorquinone and erythrosin, and thermallyactivatable molecules; 4,4' azobis(4-cyanopentanoic) acid and2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochioride. An importantcharacteristic of the initiator group is the ability to be coupled to apreformed macromer containing polymerizable groups, or to be modified toform a monomer which can take part in the macromer synthesis, which issubsequently followed by the addition of polymerizable groups.

In such an embodiment, the pendent polymerizable groups are preferablyselected from the group consisting of pendent vinyl groups, acrylategroups, methacrylate groups, ethacrylate groups, 2-phenyl acrylategroups, acrylamide groups, methacrylamide groups, itaconate groups, andstyrene groups.

In a further preferred embodiment, the polymeric backbone is selectedfrom the group consisting of synthetic macromers, such aspolyvinylpyrrolidone (PVP), polyethylene oxide L(PEO), and polyethyleneglycol (PEG); derivatizable naturally occurring polymers such ascellulose; polysaccharides, such as hyaluronic acid, dextran, andheparin; and proteins, such as collagen, gelatin, and albumin.

The macromer of the present invention can be used in a variety ofapplications, including controlled drug release, the preparation oftissue adhesives and sealants, the immobilization of cells, and thepreparation of three-dimensional bodies for implants. In one aspect, forinstance, the invention provides a method for immobilizing cells, themethod comprising the steps of combining a polymeric initiator of thepresent invention with one or more polymerizable macromers and in thepresence of cells, under conditions suitable to polymerize the macromerin a manner that immobilizes the cells.

DETAILED DESCRIPTION

As used herein the following words and terms shall have the meaningascribed below:

"macromer system" shall refer to a polymerizable polymer systemcomprising one or more polymers providing pendent polymerizable andinitiator groups. Groups can be present either on the same or differentpolymeric backbones, e.g., on either a polymerizable macromer or anon-polymerizable polymeric backbone;

"polymerizable macromer" shall refer to a polymeric backbone bearing twoor more polymerizable (e.g., vinyl) groups;

"initiator group" shall refer to a chemical group capable of initiatinga free radical reaction, present as either a pendent group on apolymerizable macromer or pendent on a separate, non-polymerizablepolymer backbone; and

"polymeric initiator" shall refer to a polymeric backbone (polymerizableor non-polymerizable) comprising one or more initiator groups andoptionally containing one or more other thermochemically reactive groupsor affinity groups.

The polymeric backbone of this invention can be either synthetic ornaturally-occurring, and includes a number of macromers previouslydescribed as useful for the preparation of polymeric matrices.Generally, the backbone is one that is soluble, or nearly soluble, inaqueous solutions such as water, or water with added organic solvent(e.g., dimethylsulfoxide) or can be rendered soluble using anappropriate solvent or combination of solvents. Alternatively, thepolymeric backbone can be a material which is a liquid under ambientphysiological conditions. Backbones for use in preparing biodegradablegels are preferably hydrolyzable under in vivo conditions.

In general, the polymeric backbones of this invention can be dividedinto two categories: biodegradable or bioresorbable, and biostablereagents. These can be further divided into reagents which formhydrophilic, hydrogel matricies and reagents which form non-hydrogelmatricies.

Bioresorbable hydrogel-forming backbones are generally naturallyoccurring polymers such as polysaccharides, examples of which include,but are not limited to, hyaluronic acid (HA), starch, dextran, heparin,and chitosan; and proteins (and other polyamino acids), examples ofwhich include but are not limited to gelatin, collagen, fibronectin,laminin, albumin and active peptide domains thereof. Matrices formedfrom these materials degrade under physiological conditions, generallyvia enzyme-mediated hydrolysis.

Bioresorbable matrix-forming backbones are generally synthetic polymersprepared via condensation polymerization of one or more monomers.Matrix-forming polymers of this type include polylactide (PLA),polyglycolide (PGA), polycaprolactone (PCL), as well as copolymers ofthese materials, polyanhydrides, and polyortho esters.

Biostable hydrogel matrix-forming backbones are generally synthetic ornaturally occurring polymers which are soluble in water, matrices ofwhich are hydrogels or water-containing gels. Examples of this type ofbackbone include polyvinylpyrrolidone (PVP), polyethylene glycol (PEG),polyacrylamide (PAA), polyvinyl alcohol (PVA), and the like.

Biostable matrix-forming backbones are generally synthetic polymersformed from hydrophobic monomers such as methyl methacrylate, butylmethacrylate, dimethyl siloxanes, and the like. These backbone materialsgenerally do not possess significant water solubility but can beformulated as neat liquids which form strong matrices upon activation.It is also possible to synthesize backbone polymers which contain bothhydrophilic and hydrophobic monomers.

Polymeric backbones of polymerizable macromers can optionally provide anumber of desirable functions or attributes, e.g., as described in theabove-captioned Hubbell patents, the disclosures of which areincorporated herein by reference. Backbones can be provided with watersoluble regions, biodegradable regions, hydrophobic regions, as well aspolymerizable regions.

As used herein, the term "polymerizable group" will generally refer to agroup that is polymerizable by initiation by free radical generation,most preferably by photoinitiators activated by visible or longwavelength ultraviolet radiation. Preferred polymerizable groups includeacrylates, methacrylates, ethacrylates, itaconates, acrylamides,methacrylamide, and styrene.

Typically, polymerizable groups are incorporated into a macromersubsequent to the initial macromer formation using standardthermochemical reactions. Thus, for example, polymerizable groups can beadded to collagen via reaction of amine containing lysine residues withacryloyl chloride or glycidyl acrylate. These reactions result incollagen containing pendent polymerizable moieties. Similarly, whensynthesizing a macromer for use as described in the present invention,monomers containing reactive groups can be incorporated into thesynthetic scheme. For example, hydroxyethylmethacrylate (HEMA) oraminopropylmethacrylamide (APMA) can be copolymerized withN-vinylpyrrolidone or acrylamide yielding a water-soluble polymer withpendent hydroxyl or amine groups. These pendent groups can subsequentlybe reacted with acryloyl chloride or glycidyl acrylate to formwater-soluble polymers with pendent polymerizable groups.

Initiator groups useful in the system of the present invention includethose that can be used to initiate, by free radical generation,polymerization of the macromers to a desired extent and within a desiredtime frame. Crosslinking and polymerization are generally initiatedamong macromers by a light-activated free-radical polymerizationinitiator. Preferred initiators for long wave UV and visible lightinitiation include ethyl eosin, 2,2-dimethoxy-2-phenyl acetophenone,other acetophenone derivatives, thioxanthone, benzophenone, andcamphorquinone.

Preferred polymeric initiators are photosensitive molecules whichcapture light energy and initiate polymerization of the macromers. Otherpreferred polymeric initiators are thermosensitive molecules whichcapture thermal energy and initiate polymerization of the macromers.

Photoinitiation of the free radical polymerization of macromers of thepresent invention will generally occur by one of three mechanisms. Thefirst mechanism involves a homolytic alpha cleavage reaction between acarbonyl group and an adjacent carbon atom. This type of reaction isgenerally referred to as a Norrish type I reaction. Examples ofmolecules exhibiting Norrish type I reactivity and useful in a polymericinitiating system include derivatives of benzoin ether and acetophenone.

The second mechanism involves a hydrogen abstraction reaction, eitherintra- or intermolecular. This initiation system can be used withoutadditional energy transfer acceptor molecules and utilizing nonspecifichydrogen abstraction, but is more commonly used with an energy transferacceptor, typically a tertiary amine, which results in the formation ofboth aminoalkyl radicals and ketyl radicals. Examples of moleculesexhibiting hydrogen abstraction reactivity and useful in a polymericinitiating system, include analogs of benzophenone, thioxanthone, andcamphorquinone.

When using a polymeric initiator of the hydrogen abstraction variety,pendent tertiary amine groups can be incorporated into the polymericbackbone of the macromer. This will insure that all free radicals formedare polymer-bound.

The third mechanism involves photosensitization reactions utilizingphotoreducible or photo-oxidizable dyes. In most instances,photoreducible dyes are used in conjunction with a reductant, typically,a tertiary amine. The reductant intercepts the induced triplet producingthe radical anion of the dye and the radical cation of the reductant.Examples of molecules exhibiting photosensitization reactivity anduseful in a polymeric initiating system include eosin Y, rose bengal,and erythrosin. Reductants can be incorporated into the polymerbackbone, thereby assuring that all free radicals will be polymer-bound.

Thermally reactive polymeric initiators are also useful for thepolymerization of macromers. Examples of thermally reactive initiatorsusable in a polymeric initiating system include 4,4'azobis(4-cyanopentanoic acid) and analogs of benzoyl peroxide.

A surprisingly beneficial effect of the use of polymeric initiators topolymerize macromers is the increased efficiency of polymerizationexhibited by these polymeric initiators as compared to their lowmolecular weight counterparts. This increased efficiency is seen in allthree photoinitiation mechanisms useful for the polymerization ofmacromers.

Polymeric initiation of monomer solutions has been investigated for itsapplication in the field of UV-curable coatings for industrial uses,c.f. U.S. Pat. No. 4,315,998 (Neckers) and PCT Application,International Publication No. WO 97/24376 (Kuester, et.al.) but therehave been no reports of the adaptation of the use of polymericinitiators for the polymerization of macromers in the presence ofbiologic material or for the creation of drug-releasing matrices.

High efficiency of initiation is particularly important in systems suchas these. It is generally desirable, when forming polymeric matrices inthe presence of biologic or bioactive materials, to minimize theexposure time of the material to the energy source used to initiatepolymerization. It is therefore imperative that the initiation systemutilized possess optimum initiation efficiency.

When matrix strength or durability are required for a particularapplication, high efficiency is again a necessary characteristic of aninitiation system. When a matrix-forming system is initiated, the freeradical polymerization of the system is propagated until gelation andvitrification of the polymerizing system render the diffusion of theelements of the matrix-forming system too difficult. Therefore, thehigher the efficiency of the initiation system, the more complete thepolymerization resulting in the formation of stronger, more durablematrices. The polymeric initiation systems described in this inventionprovide a higher degree of efficiency, without the use of accelerants,than is attainable using nonpolymer-bound, low molecular weightinitiators.

Another beneficial effect is realized when the initiating groups on thepolymeric initiators consist of groups exhibiting hydrogen abstractionreactivity, i.e., the ability to abstract hydrogens intermolecularly.The beneficial effect is important when macromer systems containingthese initiators are used as tissue adhesives, endovascular paving,formation of barriers to prevent post-surgical adhesions, or anyapplication involving the "adhesion" of the matrix to one or moresurfaces. Since initiators exhibiting this type of reactivity canabstract hydrogens from adjacent molecules, when a macromer systemcontaining polymeric initiators of this type is applied to a substrate,photoactivation of the system causes the abstraction of hydrogens fromthe substrate by the initiators, thus forming a free radical on thesubstrate and a free radical on the initiator. This diradical cansubsequently collapse forming a covalent bond between the macromersystem and the substrate.

Other initiator groups on the same macromer initiate free radicalreactions with other macromers resulting in the formation of acrosslinked matrix covalently bound to the surface. Initiator groupsexhibiting this type of reactivity include analogs of benzophenone andthioxanthone. As can be readily understood, only polymeric initiatorsare capable of accomplishing this adhesion of the matrix to a surface,low molecular weight analogs of these initiators cannot produce thisphenomenon.

In another embodiment, the polymeric initiator comprises a polymericbackbone with pendent initiator groups and pendent reactive or affinitygroups. These reactive or affinity groups enable the polymeric initiatorto bind to target groups on surfaces of interest. This allows thepolymeric initiator to bind to the surface of interest. In this manner,interfacial polymerization of macromers can be accomplished. A solutionof polymeric initiator-containing pendent reactive or affinity groups isapplied to a surface with target sites. The reactive or affinity groupson the polymeric initiator react with the sites on the surface causingthe polymeric initiator to bind to the surface. Excess polymericinitiator can then be washed away. A solution of a polymerizablemacromer is then applied to the surface. When light energy in applied tothe system, a free radical polymerization reaction is initiated only atthe surface of interest. By varying the concentration of thepolymerizable macromer and the illumination time, the thickness andcrosslink density of the resulting matrix on the surface can bemanipulated.

Generally, there are two methods by which an initiator group can beincorporated into a polymeric backbone. The first method involves theformation of a monomer which includes the initiator. This can beaccomplished readily using standard chemical reactions. For example, theacid chloride analog of an initiator can be reacted with anamine-containing monomer, to form a monomer which contains theinitiator.

The second method of incorporating initiator groups into a polymericbackbone involves coupling a reactive analog of the initiator with apreformed polymer. For example, an acid chloride analog of an initiatorcan be reacted with a polymer containing pendent amine groups forming apolymer bearing pendent initiator groups.

Polymeric matrices prepared from macromer systems can be used in avariety of applications, including:

Cellular Encapsulation. The use of hydrogels to form micro- ormacrocapsules containing cells and other tissue, is well documented inthe literature. Applications include the treatment of diabetes,Parkinson's disease, Alzheimer's disease, ALS, chronic pain, and others.Descriptions of cellular encapsulation methods can be found throughoutthe patent and scientific literature. The use of the instant inventionprovides methods of encapsulating cells in two basic ways.

1) Bulk Polymerization

In this embodiment, cellular material is mixed in a solution of themacromer system and energy subsequently added to activate initiation offree radical polymerization. Prior to initiation, the solutioncontaining the macromer system with suspended cellular material, can beplaced in molds, shaped in particular geometric shapes, or placed insidea preformed membrane system, such as a hollow fiber. Upon illuminationor other energy addition, the initiation of free radical polymerizationcauses the macromer system to gel, forming a cell-containing matrix inthe desired shape. When formed into free-standing geometric shapes, theformulation of the macromer system can be designed to provide thedesired degrees of durability and permselectivity to the subsequentlyformed matrix. When formed inside membrane structures, such as hollowfibers designed to provide the desired permselectivity, the macromersystem can be formulated to provide the desired characteristics of thecell-suspending matrix, such as biocompatibility, etc.

2) Interfacial Polymerization

In this embodiment, a membrane is formed directly on the surface of thecellular material. A solution of polymerizable or non-polymerizablepolymeric initiator-containing pendent affinity groups (e.g., positivelycharged groups) is mixed with the cellular material. The affinity groupsbind to the sites on the surface of the cellular material. The excesspolymeric initiator is subsequently washed away and the cellularmaterial suspended in a solution of polymerizable macromer. Sinceinitiator groups are present only at the surface of the cellularmaterial, when light energy is applied, polymerization is initiated onlyat the surface:macromer interface. By manipulating the duration ofillumination and macromer formulation, a polymeric matrix exhibiting thedesired characteristics of thickness, durability, permselectivity, etc.is formed directly on the surface of the cellular material.

Adhesives and sealants. Polymeric matrix systems have also foundextensive use as adhesives for tissue and other surfaces. For thisapplication, a solution of a macromer system is applied to a surface towhich adhesion is desired, another surface is contacted with thissurface, and illumination is applied forming a surface-to-surfacejunction. If a temporary adhesive is desired, the macromer system can becomposed of degradable macromers.

Barriers. Polymeric matrices can be used for the formation of barrierson surfaces for various applications. One such application is a barrierfor the prevention of tissue adhesions following surgery. For thisapplication, a macromer system in liquid form is applied to the surfaceof damaged tissue. The liquid is illuminated to polymerize themacromers. The polymeric matrix prevents other tissue from adhering tothe damaged tissue. Both degradable and non-degradable macromer systemscan be used for this purpose. As described above, both bulkpolymerization and interfacial polymerization methods can be used toprepare surface coatings of this type.

Controlled Release Carriers. Polymeric matrices find wide application ascontrolled release vehicles. For this application, a solution of amacromer system and drug, protein, or other active substance is appliedto a surface. The solution is illuminated to polymerize the macromers.The polymeric matrix contains the drug, when exposed to a physiologicalor other liquid-containing environment, the drug is slowly released intothe environment. The release profile of the entrained drug can bemanipulated by varying the formulation of the macromer system. Bothdegradable and non-degradable macromer systems can be utilized for thispurpose. Likewise, both bulk and interfacial polymerization techniquescan be used to prepare controlled drug-releasing surfaces. In analternative embodiment, a drug or other active substance can be imbibedby a preformed matrix on a surface. The absorption and releasecharacteristics of the matrix can be manipulated by varying thecrosslink density, the hydrophobicity of the matrix, and the solventused for imbibition.

Alternatively, drug-containing polymeric microspheres can be preparedusing standard techniques. A wide range of drugs and bioactive materialscan be delivered using the invention which include but are not limitedto, antithrombogenic, anti-inflammatory, antimicrobial,antiproliferative, and anticancer agents, as well as growth factors,morphogenic proteins, and the like.

Tissue Replacement/Scaffolding. Polymeric matrices have found utility asthree-dimensional scaffolding for hybrid tissues and organs. For thisapplication, a macromer system in liquid form is applied to a tissuedefect and subsequently illuminated to polymerize the macromers forminga matrix upon which ingrowing cells can migrate and organize into afunctional tissue. In one embodiment, the macromer system additionallyincludes a growth factor which is slowly released and stimulates theingrowth of desired cell types. In another embodiment, the macromersinclude pendent extracellular matrix peptides which can stimulate theingrowth of desired cell types. A third embodiment would include both ofthe above features. An alternative embodiment includes cells included inthe matrix with or without additional growth factor. The scaffolding canbe generated in vitro by placing the liquid macromer system in a mold orcavities in a device, or can be generated in vivo by applying the liquidmacromer system to a tissue defect. Both degradable and non-degradablemacromer systems could be used for this application, but degradablematrices are preferred.

Wound Dressing. Polymeric matrices have been used extensively assuperior wound dressing preparations. Currently, hydrogel andhydrocolloid wound dressing materials are being increasingly used due totheir superior wound healing properties. For this application, amacromer system in liquid form is applied to the wound site andsubsequently formed into a flexible polymeric matrix upon exposure tolight. When applied as a liquid, the macromer preparation conforms tothe irregular surface of the wound. Upon illumination, a flexible matrixis formed which is completely conformal to the surface of the wound; nofluid-filled pockets which can act as sites of bacterial infiltrationcan exist. In one embodiment, the macromer system additionally includesone or more therapeutic agents, such as growth factors or antimicrobialagents which are slowly released into the wound. Both degradable andnon-degradable macromer systems can be used for this application.

In Situ Device Formation. Polymeric materials can be implanted into thebody to replace or support the function of diseased or damaged tissues.One example of this is the use of hollow cylindrical polymeric devicesto support the structure of a coronary artery following percutaneoustransluminal coronary angioplasty (PTCA). Currently, pre-formedcylindrical devices are implanted via catheter insertion followed byballoon expansion to secure the device. The expanded device supports thestructure of the artery and prevents the reversion of the artery to theclosed position (restenosis).

For this application, a liquid macromer preparation could be applied toan injured artery via a multi-lumen catheter containing an illuminationelement. After application of the liquid macromer system to the injuredtissue, a semi-rigid polymeric matrix can be formed by a briefillumination. Upon removal of the catheter, a hollow, cylindrical,conformal polymeric device remains to support the artery and preventrestenosis. In one embodiment, the macromer system additionally includesa releasable therapeutic agent or agents, such as antiproliferativeand/or antithrombotic drugs. These agents are slowly released from theformed matrix, to provide additional therapeutic benefit to the injuredtissues. Both degradable and non-degradable macromer systems can be usedfor this application.

The invention will be further described with reference to the followingnon-limiting Examples. It will be apparent to those skilled in the artthat many changes can be made in the embodiments described withoutdeparting from the scope of the present invention. Thus the scope of thepresent invention should not be limited to the embodiments described inthis application, but only by embodiments described by the language ofthe claims and the equivalents of those embodiments. Unless otherwiseindicated, all percentages are by weight

EXAMPLES Example 1 Synthesis of 7-Methyl-9-oxothioxanthene-3-carboxylicAcid Chloride (MTA-Cl)

The 7-methyl-9-oxothioxanthene-3-carboxylic acid (MTA), 50.0 g (0.185mol), was dissolved in 350 ml of toluene and 415 ml (5.69 mol) ofthionyl chloride using an overhead stirrer in a 2 liter 3-neck roundbottom flask. N,N-Dimethylformamide (DMF), 2 ml, was added and thereaction was brought to reflux for 2 hours. After this time, the mixturewas stirred at room temperature for 16 hours. The solvent was removedunder vacuum and the product was azeotroped with 3×350 ml of toluene toremove the excess thionyl chloride. The product was recrystallized from800 ml of chloroform and the resulting solid was placed in a vacuum ovenfor 16 hours at 45° C. to complete removal of solvent. The isolatedproduct weighed 45.31 g (85% yield) and nuclear magnetic resonancespectroscopy (NMR) confirmed the desired structure. This product wasused for the preparation of a photoreactive monomer as described inExample 2.

Example 2 Synthesis ofN-[3-(7-Methyl-9-oxothioxanthene-3-carboxamido)propyl]methacrylamide(MTA-APMA)

The N-(3-aminopropyl)methacrylamide hydrochloride (APMA), 4.53 g (25.4mmol), was suspended in 100 ml of anhydrous chloroform in a 250 ml roundbottom flask equipped with a drying tube. After cooling the slurry in anice bath, the MTA-Cl, 7.69 g (26.6 mmol), was added as a solid withstirring. A solution of 7.42 ml (53.2 mmol) of triethylamine (TEA) in 20ml of chloroform was then added over a 1.5 hour time period, followed bya slow warming to room temperature. The mixture was allowed to stir 16hours at room temperature under a drying tube. After this time, thereaction was washed with 0.1 N HCl and the solvent was removed undervacuum after adding a small amount of phenothiazine as an inhibitor. Theresulting product was recrystallized from tetrahydrofuran (THE)/toluene(3/1) and gave 8.87 g (88.7% yield) of product after air drying. Thestructure of the compound was confirmed by NMR analysis.

Example 3 Preparation of N-Succinimidyl 6-Maleimidohexanoate(MAL-EAC-NOS)

6-Aminohexanoic acid, 100.0 g (0.762 moles), was dissolved in 300 ml ofacetic acid in a three-neck, 3 liter flask equipped with an overheadstirrer and drying tube. Maleic anhydride, 78.5 g (0.801 moles), wasdissolved in 200 ml of acetic acid and added to the 6-aminohexanoic acidsolution. The mixture was stirred one hour while heating on a boilingwater bath, resulting in the formation of a white solid. After coolingovernight at room temperature, the solid was collected by filtration andrinsed with 2×50 ml of hexane. After drying, the typical yield of the(Z)-4-oxo-5-aza-2-undecendioic acid was 158-165 g (90-95%) with amelting point of 160-165° C. Analysis on an NMR spectrometer wasconsistent with the desired product.

(Z)-4-Oxo-5-aza-2-undecendioic acid, 150.0 g (0.654 moles), aceticanhydride, 68 ml (73.5 g, 0.721 moles), and phenothiazine, 500 mg, wereadded to a 2 liter three-neck round bottom flask equipped with anoverhead stirrer. Triethylamine, 91 ml (0.653 moles), and 600 ml of THFwere added and the mixture was heated to reflux while stirring. After atotal of 4 hours of reflux, the dark mixture was cooled to <60° C. andpoured into a solution of 250 ml of 12 N HCl in 3 liters of water. Themixture was stirred 3 hours at room temperature and then was filteredthrough a Celite 545 pad to remove solids. The filtrate was extractedwith 4×500 ml of chloroform and the combined extracts were dried oversodium sulfate. After adding 15 mg of phenothiazine to preventpolymerization, the solvent was removed under reduced pressure. The6-maleimidohexanoic acid was recrystallized from hexane/chloroform (2/1)to give typical yields of 76-83 g (55-60%) with a melting point of81-85° C. Analysis on a NMR spectrometer was consistent with the desiredproduct.

The 6-maleimidohexanoic acid, 20.0 g (94.7 mmol), was dissolved in 100ml of chloroform under an argon atmosphere, followed by the addition of41 ml (0.47 mol) of oxalyl chloride. After stirring for 2 hours at roomtemperature, the solvent was removed under reduced pressure with 4×25 mlof additional chloroform used to remove the last of the excess oxalylchloride. The acid chloride was dissolved in 100 ml of chloroform,followed by the addition of 12.0 g (0.104 mol) of N-hydroxysuccinimideand 16.0 ml (0.114 mol) of triethylamine. After stirring overnight atroom temperature, the product was washed with 4×100 ml of water anddried over sodium sulfate. Removal of solvent gave 24.0 g (82%) ofMAL-EAC-NOS which was used without further purification. Analysis on anNMR spectrometer was consistent with the desired product.

Example 4 Preparation of a Copolymer of MTA-APMA, MAL-EAC-NOS, andN-Vinylpyrrolidone

A polymeric initiator is prepared by copolymerization of a monomercharge consisting of 5 mole % MTA-APMA, 10 mole % MAL-EAC-NOS, and 85mole % N-vinylpyrrolidone (VP). The polymerization is run in formamideor other suitable solvent using 2,2'-azobisisobutyronitrile (AIBN) as aninitiator and N,N,N',N'-tetramethylethylenediamine (TEMED) as an oxygenscavenger. Mercaptoethanol is added as a chain transfer reagent at aconcentration designed to give a molecular weight between 2,000 and20,000 daltons. Upon completion of the polymerization, the copolymer isprecipitated by addition of ether or other non-solvent for the polymer.After isolation by filtration, the product is washed extensively withthe precipitating solvent to remove residual monomers and low molecularweight oligomers. The copolymer is dried under vacuum and is storeddesiccated to protect the hydrolyzable N-oxysuccinimide (NOS) esters.

Example 5 Synthesis of a Photoreactive Macromer Derived from aPoly(caprolactone-co-lactide) Derivative of Pentaerythritol Ethoxylate

A 15 gram scale reaction was performed by charging a thick-walled tubewith 8.147 g (56.5 mmol) of 1-lactide(3,6-dimethyl-1,4-dioxane-2,5-dione) and 6.450 g (56.5 mmol) ofε-caprolactone. To this mixture was added 0.402 g (1.49 mmol) ofpentaerythritol ethoxylate (ave. MW appprox. 270) to providepolymerization sites and control molecular weight. This mixture waswarmed gently until dissolution of all reagents was complete. Thecatalyst, stannous 2-ethylhexanoate (0.015 ml) was added and thereaction vessel sealed. The reaction mixture was warmed to 150° C. andstirred for 20 hours. The resulting polymer was dissolved in chloroformand dialyzed against methanol using 1000 MWCO dialysis tubing. Afterdialysis, the solvent was removed in vacuo. The purified polymer wasdissolved in chloroform and treated with 2.41 g (23.8 mmol) of TEA. Tothis reaction mixture was added 292 mg (1.19 mmol) of 4-benzoylbenzoylchloride (BBA-Cl) and the resulting mixture was stirred for 16 hours. Tothis reaction mixture was added 0.734 g (8.11 mmol) of acryloyl chlorideand the reaction was stirred an additional 8 hours. The modified polymerwas purified by dialysis against methanol using 1000 MWCO dialysistubing. After dialysis, the solvent was removed in vacuo and the polymer(15.36 grams) stored desiccated at room temperature.

Example 6 Synthesis of Water Soluble Siloxane Macromer with PendentInitiator Groups

Fifty grams of a water-soluble siloxane macromer with pendent initiatorgroups were synthesized by first dissolving 50 grams of commerciallyavailable poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]-graft-poly(ethylene glycol) 3-aminopropylether (Aldrich Chemical) in 50 ml of methylene chloride. To thissolution was added 5.0 g (49 mmol) of TEA. The reaction solution wascooled to -50° C., then transferred to a stir plate at room temperature.MTA-Cl, 1.0 g (3.5 mmol), prepared according to the general method inExample 1, and 5.0 g (55 mmol) of acryloyl chloride were added and thesolution was stirred for 6 hours at room temperature. The solution wasdialyzed against deionized water using 3500 MWCO dialysis tubing and thewater was subsequently removed in vacuo. The product (48.4 grams) wasstored desiccated at room temperature.

Example 7 Synthesis of a Polymerizable Hyaluronic Acid

Two grams of hyaluronic acid (Lifecore Biomedical, Chaska, MN) weredissolved in 100 ml of dry formamide. To this solution were added 1.0 g(9.9 mmol) of TEA and 4.0 g (31 mmol) of glycidyl acrylate. The reactionmixture was stirred at 37° C. for 72 hours. After exhaustive dialysisagainst deionized water using 12-14k MWCO dialysis tubing, the product(2.89 grams) was isolated by lyophilization.

Example 8 Preparation of a Photoderivatized Polyacrylamide (Photo-PAA)

Acrylamide, 10.24 g (0.144 mol), was dissolved in 200 ml of deionizedwater. To the solution was added 0.279 g (1.56 mmol) of APMA, 0.33 g(1.45 mmol) of ammonium persulfate and 0.155 g (1.33 mmol) of TEMED. Thesolution was evacuated in a filter flask with a water aspirator for 10minutes. The tubing was clamped and the solution left under vacuum forone hour. The resulting polymer solution was dialyzed against deionizedwater using 12-14k MWCO dialysis tubing. To 150 ml of polymer solutionin a PTFE bottle containing 3.0 grams of polymer was added 0.504 ml(3.62 mmol) of TEA. To this solution was added 30 ml of 28.4 mg/ml (3.48mmol) 4-benzoylbenzoyl chloride in CHCl₃. The bottle was capped tightlyand shaken for one hour. The bottle was then centrifuged for 10 minutesto separate the phases after which the aqueous layer was removed,dialyzed against deinoized water using 12-14k MWCO dialysis tubing, andlyophilized. The product (3.21 grams) was stored, dessicated at roomtemperature.

Example 9 Synthesis of the N-Hydroxysuccinimide Ester of Eosin Y

Eosin Y, 1.00 g (1.54 mmol), was dissolved in 10 ml dry dioxane withstirring, gentle warming and some sonication. After the solution wascomplete, the orange solution was cooled to room temperature underargon. N-Hydroxysuccinimide, 0.195 g (1.69 mmol), and1,3-dicyclohexylcarbodiimide, 0.635 g (3.08 mmol), were added as solids.The resulting red mixture was stirred at room temperature for 48 hoursunder an inert atmosphere. After this time the solid was removed byfiltration and washed with dioxane. The filtrate was concentrated invacuo to give 1.08 g (94% yield) of a glassy red solid.

Example 10 Synthesis of a Copolymer of APMA, Methyl Methacrylate, andN-Vinylpyrrolidone Followed by Addition of Acryloyl Groups

The following ingredients for the copolymer were placed in a glassvessel and dissolved in 20 ml DMSO: APMA (2.68 g, 15.0 mmol), VP (6.74ml, 63.1 mmol), methyl methacrylate (mMA) (0.334 ml, 3.12 mmol),mercaptoethanol (0.053 ml, 0.76 mmol), AIBN (0.041 g, 0.25 mmol), andTEMED (0.057 ml, 0.38 mmol). After solution was complete, the monomersolution was degassed, blanketed with argon and placed in an agitatingincubator at 55° C. The copolymer was dialyzed against deionized waterin 6-8,000 MWCO dialysis tubing. The dialyzed solution (˜400 ml) wasloaded with acrylate groups. TEA, 5.0 ml (35.9 mmol), was added withstirring. The solution was placed in a freezer for 5-10 minutes to cool.After this time, 5.0 ml (61.5 mmol) of acryloyl chloride in 5 ml ofchloroform were added with stirring. The reaction mixture was stirred atroom temperature for 16 hrs. After this time the acrylated polymer wasdialyzed against deionized water using 6-8,000 MWCO tubing. The productwas lyophilized and 7.10 g were obtained.

Example 11 Synthesis of a Copolymer of MTA-APMA, APMA, MethylMethacrylate, and N-Vinylpyrrolidone Followed by Addition of AcryloylGroups

The following ingredients for the copolymer were placed in a glassvessel and dissolved in 20 ml DMSO: MTA-APMA (0.613 g, 1.55 mmol), APMA(2.578 g, 14.4 mmol), VP (6.27 ml, 58.7 mmol), mMA (0.319 ml, 2.98mmol), mercaptoethanol (0.054 ml, 0.77 mmol), AIBN (0.039 g, 0.24 mmol),and TEMED (0.053 ml, 0.35 mmol). After solution was complete, themonomer solution was degassed, blanketed with argon and placed in anagitating incubator at 55° C. The copolymer was dialyzed againstdeionized water in 6-8,000 MWCO dialysis tubing. The dialyzed solutionwas protected from light and loaded with acrylate groups. TEA, 5.0 ml(35.9 nunol), was added with stirring. The solution was placed in afreezer for 5-10 minutes to cool. After this time, 5.0 ml (61.5 mmol) ofacryloyl chloride in 5 ml of chloroform were added with stirring. Thereaction mixture was stirred at room temperature for 9 hrs. After thistime the acrylated polymer was dialyzed against deionized water using6-8,000 MWCO tubing and protected from light. The product (8.88 grams)was isolated by lyophilization.

Example 12 Evaluation of Matrix Formation

A 15% solution of the co-polymer from Example 11 was prepared in 10%DMSO/water. The MTA content of the solution was estimated by measuringthe absorbance of the solution at 395 nm(A@395 nm=42.6). A 15% solutionof the co-polymer from Example 10 (same co-polymer as that described inExample 11 but with no MTA-APMA) was prepared in 10% DMSO/water. MTA wasadded to this solution until its absorbance at 395 nm matched that ofthe solution described above. The two solutions were identical inconcentration of co-polymer and photoinitiator, the only differencebetween them being that in one solution the photoinitiator was presentin polymeric form(POLY) and in the other the photoinitiator was presentin non-polymeric form(NON).

In order to compare the matrix forming ability of the two solutions thefollowing evaluation was undertaken: the indentations in the lid of a 96well microtiter plate were used as miniature molds to evaluate theability of the photoreactive polymer solutions to form solid hydrogeldiscs upon illumination. The indentations are eight millimeters indiameter and approximately 0.6 millimeters deep. 30 microliters ofpolymer solution will just fill the indentatation. Thirty microliters ofboth the (POLY) and (NON) solutions were added to wells. After additionof the polymer solutions, the lids were illuminated using an EFOSUltracure 100 SS illumination system equipped with a 400-500 nm filter,for varying lengths of time. After illumination the lid was flooded withwater and each polymer formulation rated for its ability to form soliddiscs using the following arbitrary scale:

0=liquid, no gelation

1=soft gel, unable to remove from mold

2=firm gel, removable from mold with slight difficulty

3=very firm gel, easily removed from mold

4=very firm gel, elastomeric properties evident

Results:

    ______________________________________                                               Time(sec)                                                              Polymer  2     5         10  30      60  120                                  ______________________________________                                        (POLY)   1     2         3   4       4   4                                      (NON)             0    0    1    2    3    3                                       Matrix formation                                                       ______________________________________                                    

The polymer solution containing the polymer-bound initiator(POLY) formedmatrices more rapidly and more completely than the polymer solutioncontaining non-polymer-bound initiator(NON) when exposed to lightenergy.

Example 13 Synthesis of an Eosin Substituted Polymer

N-Vinylpyrrolidone, 10.0 g (90.0 mmol), was dissolved in 50 ml DMSO. Tothe solution was added 0.30 g (1.68 mmol) of APMA, 0.15 g (0.91 mmol) ofAIBN, and 0.10 g (0.86 mmol) of TEMED. The solution was sparged withnitrogen for 20 minutes and incubated at 55° C. for 20 hours. Theresulting polymer was purified by dialysis against water and isolated bylyophilization.

Three grams of the polymer were dissolved in 150 mls dry dioxane. Tothis solution was added 0.504 ml (3.62 mmoles) of TEA. Subsequently,2.74 grams (3.5 mmoles) of the N-hydroxysuccinimide ester of Eosin Y wasadded and the reaction mixture stirred for two hours at roomtemperature. The solution was dialyzed against dH₂ O using 12-14 kdacut-off dialysis tubing and lyophilized to isolate the product. Thereaction yielded 3.96 grams of red polymer.

Example 14 A Biodegradable Tissue Adhesive

A solution was prepared consisting of 5% polymerizable hyaluronic acid(Example 7) and 2% photoderivatized polyacrylamide (Example 8) in water.This reagent was evaluated for use as a tissue adhesive using cellulosedialysis tubing as a tissue model.

Shear strength testing was performed on dialysis tubing. The tubing wasslit and cut into 2 cm×4 cm pieces. The pieces were soaked in waterbriefly, removed, and tested while still damp. One piece was laid flaton a surface and 10 μl of adhesive applied to one end of the strip.Another piece was laid over this piece with a 1 cm overlap betweenpieces. When evaluating the photoactivatable adhesive (2/5 HA), theoverlap area was illuminated for 10 seconds. When evaluating a controladhesive, the adhesive was allowed to set for five minutes. The bondedsamples were mounted in a tensiometer lengthwise by the ends such thatthe plane of the area of adhesive was parallel to the axis of thetensiometer. The samples were extended at the rate of 1 cm/minute untiladhesive or substrate failure, and the force at failure recorded.Substrate-only, and, for photoactivatable adhesive, non-illuminatedsamples, were included as controls in the evaluations.

    ______________________________________                                                 Maximum Force                                                                             Adhesive Failed                                                                           Substrate Failed                               Adhesive Generated Kg     Before Substrate   Before Adhesive                ______________________________________                                        2/5 HA   0.53        0/4         4/4                                            2/5 HA (no               0.081             4/4             0/4                illumination)                                                                 Fibrin glue              0.045             4/4             0/4                Cyanoacrylate            0.49              0/4             4/4              ______________________________________                                    

Example 15 Formation of an in situ Hydrogel Wound Dressing

Photopolymerizable, matrix-forming reagents were evaluated for efficacyas in situ wound dressings.

Preparation of reagents:

An experimental in situ forming wound dressing was prepared by:

1) Dissolving reactive macromer from Example 10 at 20% into a sterile 6%glycerin solution in water.

2) Preparing a sterile solution of polymeric eosin reagent from Example12 at 4% in water and a sterile solution of 2M triethanolamine (TEA) inwater.

3) Transporting the three sterile solutions to a surgical suite forapplication to wound sites created on porcine skin.

Four young female China White swine weighing between 15-20 kg wereanesthetized and 12 wounds inflicted on one side of each pig. Woundswere 1"×2" and 0.015" deep and were inflicted by a calibratedelectrodermatome (Padgett). The wounds were inflicted in two rows of sixon the thoracic and paravertebral area of each pig, leavingapproximately two inches between adjacent wounds. The wounds wererandomized and received one of three treatments:

1) No treatment (control)

2) Application of OpSite®, a semi-occlusive wound dressing from Smithand Nephew, Inc.

3) Experimental photo-curable dressing

To apply the experimental dressing, 0.5 mls of the polymeric-eosinsolution and 0.5 mls of the TEA solution were added to themacromer/glycerin solution yielding a photo-wound dressing solution. Thesolution was transferred to 16 three ml sterile syringes (2 ml/syringe)and one syringe was used to application to each wound site. Thesolutions were applied to each assigned wound site (approximately 1.5mls solutions/site) and allowed to flow over the site. The solutionswere fixed by illumination with a 150 W incandescent light bulbpositioned four inches from the wound surface for 30 seconds. Thedressing solution readily formed into a durable, rubbery hydrogel whichadhered very well to the wound sites. Sterile 4×4 gauze pads were placedover the entire wounded area of each pig, and the pigs placed in sterilestockinettes. On selected days (3, 4, 5, and 7), one pig was euthanizedand the effect of dressing on wound epithelialization and repairevaluated. Evaluation of effect of dressing on wound epithelializationand repair:

Following euthanasia, skin wounds were removed from the underlying deepsubcutaneous tissue and fixed in 10% neutral buffered formalin solution.After fixation, five biopsy sites from each wound were obtained with a 6mm Keys skin biopsy punch. Each biopsy was packaged, labeled andsubmitted for histological sectioning. Histological sections weresectioned at 4 microns and stained with hematoxylin and eosin.Histological sections were examined with the microscope without knowingthe type of covering placed over the wound site. The following criteriawere evaluated and scored in microscopic examination:

Degree of epithelialization of the wound

Magnitude of the inflammatory reaction

Degree of fibroplasia in the wound

Degree of damage to subcutaneous tissue:

Morphometric analysis of cell types in the histological sections wereused to help differentiate the degree of inflammatory reaction present.The number of polymorphonuclear cells, lymphocytic cells, andfibroblasts was evaluated. Each histological biopsy was graded on ascale of 1-5.

Degree of Inflammatory Reaction:

1. No or borderline cellular inflammatory reaction

2. Minimal inflammation

3. Moderate density of inflammatory cells with some exudate

4. Severe, high density of inflammatory cells in or on the wound tissuewith thicker layer of exudate

5. Excessive inflammation, with signs of dense foci of inflammatorycells infiltrating the wound tissue or on the wound and forming a thicklayer of inflammatory exudate.

Degree of Wound Epithelialization:

1. Stratum coreum present at least 4 layers of cells and entireepidermal surface is present.

2. Stratum comeum is present at least 1 layer of cells and entireepidermal surface is present.

3. Stratum corneum is present at least 1 layer of cells and 1/2 ofepidermal surface is covered.

4. No stratum comeum is present; minimal inflammation of thesubepidermal tissue.

5. No stratum corneum is present; moderate inflammation in subepidermaltissue.

Degree of Fibroplasia in the Wound:

1. No fibroplasia in the wound

2. Mild fibroplasia in the wound involving 1/3 to 1/2 wound surface

3. Mild fibroplasia in the wound involving 2/3 or more of the wound

4. Moderate fibroplasia involving 1/3 to 1/2 of the wound

5. Severe fibroplasia involving 1/2 or more of the wound

Degree of Damage to the Subcutaneous Tissue:

1. No damage to the subcutaneous tissue

2. Mild damage to the subcutaneous tissue with mild edema and fewinflammatory cells.

3. Moderate damage to the subcutaneous tissue with moderate edema andmoderate accumulation of inflammatory cells

4. Severe damage to the subcutaneous tissue with severe edema and largenumber of inflammatory cells

5. Excessive damage to the subcutaneous tissue with dense foci ofinflammatory cells

Results:

Each biopsy was graded blindly using the criteria listed above. When thehistological examination was completed, the graded biopsies werecorrelated with the wound sites. A single average score for eachdressing was calculated by adding all the scores for every site for eachdressing and dividing by the number or scores.

The total scores for each type of wound dressing on days 3, 4, 5, and 7were evaluated with an ANOVA SAS program for data intervals tostatistically evaluate if there was any difference between the threetypes of wound treatments administered. Only two scores were found to bestatistically significant:

1. On day 4 following wound creation the mean for the OpSite® dressingwas 2.4 and was found to be statistically significant when compared tothe control and experimental wound sites.

2. On day 7 following the creation of the wounds the mean for theexperimental dressing, 1.8 was found to be statistically significantwhen compared to the control and the OpSite® wound dressings.

On day 7 post-wound creation, the wound sites treated with theexperimental photocurable dressing showed significantly superior healingto those that were untreated or treated with OpSite® dressing, as judgedby the criteria described.

Example 16 A Bioresorbable Drug Delivery Coating

A solution of 33% of the macromer from Example 5 was prepared inethanol. Ten centimeter lengths of polyurethane rod (PU) were dippedinto the macromer solutions and illuminated for six minutes to form amatrix. This procedure resulted in the formation of a very durable,tenacious, and flexible coating on the rod. One gram of chlorhexidinediacetate (an antimicrobial agent) was dissolved in 10 mls of themacromer solution and the coating process repeated on additional PUrods. This also resulted in a tenacious, durable, and flexible coatingon the rods. The rods were cut into one centimeter pieces and evaluatedin a zone of inhibition analysis.

Coated dye-containing pieces, coated no-drug controls, and uncoatedpieces were placed in Mueller-Hinton agar plates which were swabbed witha 10⁶ suspension of Staphylococcus epidermidis (Christensen RP62A).These pieces functioned as unwashed controls and were transferred tofreshly swabbed agar plates each day for 60 days.

Additional pieces, no-drug controls (both coated and uncoated) anddrug-incorporated coated, wer placed in snap-cap vials and washed with50% Normal Calf Serum in PBS. The tubes were placed on an orbital shakerand incubated at 37° C. and 200 rpm for 20 days. Each day the washsolution was removed and replaced with fresh solution. Periodically,pieces were removed from the serum/PBS and placed in agar as describedabove. Zones of inhibition resulting from these pieces were recorded andcompared to the zones produced by unwashed pieces.

The no-drug coated control pieces, both coated and uncoated, produced nozones. On day 0, both washed and unwashed drug-incorporated piecesproduced zone of 24.5 mm. On day 20, when the final washed pieces wereevaluated, the unwashed pieces were producing zones of 17.5 mm, and thewashed pieces were producing zones of 9.5 mm. On day 60, when theexperiment was terminated, the unwashed pieces were still producingzones of 17 mm.

This experiment demonstrates the utility of this matrix-forming polymerat producing drug delivery coatings which provide a long-term deliveryof a bioactive agent.

Example 17 A Biostable Drug Delivery Coating

A solution of 25% of the macromer from Example 6 was prepared in 50%IPA/H₂ O. Ten centimeter lengths of polyurethane rod were dipped intothe macromer solution and illuminated for six minutes to form matrix.This procedure resulted in the formation of a very durable, tenacious,and flexible coating on the rod. Five hundred milligrams ofchlorhexidase diacetate was dissolved in 10 mls ethanol. Half of thecoated rods were soaked in this solution for 60 minutes at roomtemperature, and half of the rods were soaked in neat ethanol under thesame conditions. After soaking, the rods were removed from the ethanoland allowed to dry for 20 hours at room temperature. The rods were cutinto one centimeter pieces and evaluated in a zone of inhibitionanalysis.

Uncoated control, coated control, and coated drug-incorporated pieceswere placed in Mueller-Hinton agar plates which were swabbed with a 10⁶suspension of Staphylococcus epidermidis (Christensen RP62A). Theseplates were incubated for 20 hours at 37° C. The zone where no bacterialgrowth was evident around each piece was measured and the piecetransferred to a freshly swabbed agar plate each day for 14 days.

The uncoated control pieces and the coated control pieces produced nozones. On day 0, the drug-incorporated coated pieces produced averagezones of 25 mm. These pieces continued to produce zones each day. On day14, when the experiment was terminated, the pieces produced averagezones of 6 mm.

Example 18 Formation of a Three-Dimensional Device

One end of a 3 mm diameter teflon-coated rod was dipped to a level of1.5 cm in neatBBA-acryloylpolytetra(caprolactone-f&-lactide)pentaerthritol ethoxylate(see Example 5) and immediately illuminated, with rotation, for 10seconds suspended between opposed Dymax lamps. After illumination, asemi-rigid elastomeric coating had formed on the rod. The rod was cooledto facilitate removal of the polymeric coating. The closed end of thecylinder was removed with a razor blade, thus forming a hollowcylindrical device of 1.25 cm in length and 3.5 mm in diameter.

Example 19 Synthesis of a Polymerizable Collagen

One gram of soluble collagen (Semed-S, Kensey-Nash Corp.) (a mixture ofTypes I and III) was dissolved in 50 mls of 0.01 N HCl. When dissolved,1.25 gms triethylanmine (12.4 mmoles) was added to the reaction mixture.One gram of acryloyl chloride (11.0 mmoles) dissolved in one milliliterof methylene chloride was added to the reaction vessel and the mixturewas stirred for 20 hours at room temperature.

The reaction mixture was dialyzed exhaustively against dH₂ O, and theproduct isolated by lyophilization. A yield of 1.17 grams ofpolymerizable collagen was realized.

Example 20 A Collagen Scaffolding that Contains a Bone MorphogenicProtein

A. Preparation of the Solidified Scaffolding.

A solution of liquid macromer is prepared which consists of 5% (w/v) ofpolymerizable collagen (Example 19) plus 1% (w/v) of photoderivatizedpolyacrylamide (prepared as described in Example 8) in phosphatebuffered saline, pH 7.4. To this is added 50 μg/ml (0.005% w/v) of bonemorphogenic protein (13MP-7 from a private source). Aliquots of theabove solution (150 μl) are then placed in molds (8 mm diameter and 3 mmhigh) and are illuminated for 10 seconds with a Dymax lamp (as describedin Example 13) to solidify the collagen scaffolding. Control disks ofsolidified collagen scaffolding are prepared via the same protocolexcept that BMP-7 is not added.

B. Evaluation of the Solidified Scaffolding.

Disks of solidified collagen scaffolding with BMP-7 are evaluated forstimulation of bone growth in a rat cranial onlay implant model. In thismodel, the periosteal membrane is removed and the collagen disks areimplanted on the cranium. After 30 days, the implants and adjacentcranial bone are removed, fixed in cold methanol, embedded in PMMA,sectioned, ground to 50-100 μm thickness, stained with Sandersons RapidBone Stain, and counterstained with Van Gieson's picro-fuchsin. Thisprotocol evaluates nondecalcified bone, with mature bone staining red,immature bone staining pink, cartilage staining blue-gray, andundegraded collagen appearing acellular and pale yellow.

One control consists of disks of solidified collagen scaffolding lackingBMP-7. A second control consists of 150 μl of nonilluminated liquidmacromer solution which contains BMP-7 (the same solution compositionthat was placed in molds and illuminated to produce the solidifiedcollagen scaffolding containing BMP-7).

When evaluated histologically at 30 days as described above, theexperimental disks (solidified collagen scaffolding containing BMP-7)show extensive bone formation in the space originally occupied by thecollagen disk. In contrast, both controls (the solidified collagenscaffolding lacking BMP-7 and the nonilluminated liquid control solutioncontaining BMP-7) show little or no bone formation. The amount of bonethat forms with the controls is less than 25% of that observed with theexperimental disks, therefore demonstrating that the solidified collagenscaffolding greatly enhances BMP-stimulated bone formation.

What is claimed is:
 1. A crosslinkable macromer system comprising one ormore polymers providing pendent polymerizable and pendent initiatorgroups wherein the system is adapted to be polymerized in order to forma matrix suitable for iin vivo application, and wherein either:(a) thepolymerizable groups and initiator group(s) are pendent on differentpolymers and the initiator groups are independently selected from thegroup consisting of long wave ultraviolet activatable molecules selectedfrom the group consisting of benzophenone, thioxanthones, and benzoinethers; visible light activatable molecules selected from the groupconsisting of ethyl eosin, eosin Y, rose bengal, camphorquinone anderythrosin; and thermally activatable molecules selected from the groupconsisting of 4,4' azobis(4-cyanopentanoic) acid, and2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride, and benzoylperoxide; and the pendent polymerizable groups are selected from thegroup consisting of vinyl groups, acrylate groups, methacrylate groups,ethacrylate groups, 2-phenyl acrylate groups, acrylamide groups,methacrylamide groups, itaconate groups, and styrene groups, or (b) thepolymerizable groups and the initiator group(s) are pendent on the samepolymer and the initiator groups are independently selected from thegroup consisting of long wave ultraviolet activatable molecules selectedfrom the group consisting of thioxanthones, and benzoin ethers; visiblelight activatable molecules selected from the group consisting of ethyleosin, eosin Y, rose bengal, camphorquinone and erythrosin; andthermally activatable molecules selected from the group consisting of4,4' azobis(4-cyanopentanoic) acid, and 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and benzoyl peroxide; and the pendentpolymerizable groups are selected from the group consisting of vinylgroups, acrylate groups, methacrylate groups, ethacrylate groups,2-phenyl acrylate groups, acrylamide groups, methacrylamide groups,itaconate groups, and styrene groups; or (c) the polymerizable groupsand the initiator group(s) are pendent on the same polymer and theinitiator groups are independently selected from the group consisting oflong wave ultraviolet activatable molecules selected from the groupconsisting of benzophenone, thioxanthones, and benzoin ethers; visiblelight activatable molecules selected from the group consisting of ethyleosin, eosin Y, rose bengal, camphorquinone and erythirosin; andthermally activatable molecules selected from the group consisting of4,4' azobis(4-cyanopentanoic) acid, and 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and benzoyl peroxide; and the pendentpolymerizable groups are selected from the group consisting of acrylategroups, methacrylate groups, ethacrylate groups, 2-phenyl acrylategroups, acrylamide groups, methacrylamide groups, itaconate groups, andstyrene groups.
 2. A macromer system according to claim 1 wherein thependent initiator groups are bonded to one or more monomers used toprepare the polymer.
 3. A macromer system according to claim 1 whereinthe pendent initiator groups are bonded to the polymer itself.
 4. Amacromer system according to claim 1 wherein the system comprisespolymers bearing both initiator groups and polymerizable groups incombination with polymers providing only pendent polymerizable groups.5. A macromer system according to claim 1 wherein photoinitiation occursby means of a hydrogen abstraction reaction.
 6. A macromer systemaccording to claim 5 wherein the system further comprises one or morepolymer-pendent energy transfer acceptor molecules.
 7. A macromer systemaccording to claim 6 wherein the energy transfer molecule is selectedfrom the group consisting of tertiary amines.
 8. A macromer systemaccording to claim 1 wherein the groups are pendent upon polymersselected from the group consisting of synthetic polymers andnaturally-occurring polymers.
 9. A macromer system according to claim 8wherein the polymer is selected from the group consisting ofbiodegradable polymers and biostable polymers.
 10. A macromer systemaccording to claim 9 wherein the polymer is selected from the groupconsisting of polymers adapted to form hydrogel matricies and polymersadapted to form non-hydrogel matrices.
 11. A macromer system accordingto claim 10 wherein the polymer comprises a biodegradable polymeradapted to form hydrogel matrices.
 12. A macromer system according toclaim 11 wherein the polymer is a naturally-occurring polymer selectedfrom the group consisting of polysaccharides and polyamino acids.
 13. Amacromer system according to claim 12 wherein the polysaccharides areselected from the group consisting of hyaluronic acid, starch, dextran,heparin, and chitosan, and the polyamino acids are selected from thegroup consisting of gelatin, collagen, fibronectin, laminin, albumin andactive peptide domains thereof.
 14. A macromer system according to claim8 wherein the polymer comprises a synthetic polymer prepared viacondensation polymerization of one or more monomers.
 15. A macromersystem according to claim 9 wherein the polymers are hydrophobic,biodegradable polymers selected from the group consisting ofpolylactides, polyglycolides, polycaprolactones, and copolymers of anycombination of lactides, glycolides and caprolactones, and furtherconsisting of polyanhydrides and polyortho esters.
 16. A macromer systemaccording to claim 9 wherein the polymers are biostable, hydrophilicpolymers selected from the group consisting of polyvinylpyrrolidone,polyethylene glycol, polyacrylamide, and polyvinyl alcohol.
 17. Amacromer system according to claim 9 wherein the polymers are synthetic,biostable polymers formed by the polymerization of hydrophobic monomersselected from the group consisting of methyl methacrylate, butylmethacrylate, and dimethyl siloxanes.
 18. A macromer system according toclaim 1 wherein the polymers further comprise pendent reactive groups oraffinity groups.
 19. A macromer system according to claim 18 herein thereactive groups or affinity groups are adapted to bind to target groupson a surface of interest.
 20. A method of forming a polymeric matrixadapted for in vivo application, the method comprising the steps ofproviding a macromer system according to claim 1, applying the system toa substrate, and cross-linking the system by free radical polymerizationto form a matrix.
 21. A method according to claim 20, wherein the invivo application is selected from the group consisting of cellularencapsulation, adhesives/sealants, barriers, controlled releasecarriers, tissue replacement, wound dressing, and in situ deviceformation.
 22. A method according to claim 20 wherein photoinitiation ofthe system occurs by means of a hydrogen abstraction reaction.
 23. Amethod according to claim 22 wherein the system further comprises one ormore polymer-pendent energy transfer acceptor molecules.
 24. A methodaccording to claim 23 wherein the energy transfer acceptor molecules areselected from the group consisting of tertiary amines.
 25. A methodaccording to claim 22 wherein photoinitiation results in the formationof covalent bonds between the matrix and the substrate.
 26. A polymericmatrix adapted for in vivo application the matrix comprising a macromersystem according to claim 1, the system having bean applied to asubstrate and cross-linked by free radical polymerization to form amatrix.
 27. A matrix according to claim 26, wherein the in vivoapplication is selected from the group consisting of cellularencapsulation, adhesives/sealants, barriers, controlled releasecarriers, tissue replacement, wound dressing, and in situ deviceformation.
 28. A matrix according to claim 26 wherein photoinitiation ofthe system occurs by means of a hydrogen abstraction reaction.
 29. Amatrix according to claim 28 wherein the system further comprises one ormore polymer-pendent energy transfer acceptor molecules.
 30. A matrixaccording to claim 29 wherein the energy transfer acceptor molecules areselected from the group consisting of tertiary amines.
 31. A matrixaccording to claim 28 wherein photoinitiation results in the formationof covalent bonds between the matrix and the substrate.
 32. A macromersystem according to claim 1 wherein the thioxanthones are selected fromthe group consisting of [(9-oxo-2-thioxanthanyl)-oxy]acetic acid and2-hydroxy thioxanthone, and the benzoin ethers comprisevinyloxymethylbenzoin methyl ether.
 33. A method according to claim 20wherein the thioxanthones are selected from the group consisting of[(9-oxo-2-thioxanthanyl)-oxy]acetic acid and 2-hydroxy thioxanthone, andthe benzoin ethers comprise vinyloxymethylbenzoin methyl ether.
 34. Apolymeric matrix according to claim 26 wherein the thioxanthones areselected from the group consisting of[(9-oxo-2-thioxanthanyl)-oxy]acetic acid and 2-hydroxy thioxanthone, andthe benzoin ethers comprise vinyloxymethylbenzoin methyl ether.
 35. Amethod of forming a polymeric matrix adapted for in vivo application,the method comprising the steps of providing a macromer system accordingto claim 1, applying the system to a substrate, and cross-linking thesystem by free radical polymerization to form a matrix, whereinphotoinitiation of the system occurs by means of a hydrogen abstractionreaction, and wherein photoinitiation results in the formation ofcovalent bonds between the matrix and the substrate.
 36. A polymericmatrix adapted for in vivo application, the matrix comprising a macromersystem according to claim 1, the system having been applied to asubstrate and cross-linked by free radical polymerization to form amatrix, wherein photoinitiation of the system occurs by means of ahydrogen abstraction reaction, and wherein photoinitiation results inthe formation of covalent bonds between the matrix and the substrate.37. A polymeric matrix adapted for in vivo application, the matrixcomprising a macromer system according to claim 1, the system havingbeen applied to a substrate and cross-linked by free radicalpolymerization to form a matrix, wherein photoinitiation of the systemoccurs by means of a hydrogen abstraction reaction, and wherein thethioxanthones are selected from the group consisting of[(9-oxo-2-thioxanthanyl)-oxy]acetic acid and 2-hydroxy thioxanthone, andthe benzoin ethers comprise vinyloxymethylbenzoin methyl ether.
 38. Acrosslinkable macromer system comprising one or more polymers providingpendent polymerizable and pendent initiator groups wherein the system isadapted to be polymerized in order to form a matrix suitable for in vivoapplication, and wherein the initiator groups are independently selectedfrom the group consisting of long wave ultraviolet activatable moleculesselected from the group consisting of benzophenone, thioxanthones, andbenzoin ethers; visible light activatable molecules selected from thegroup consisting of ethyl eosin, eosin Y, rose bengal, camphorquinoneand erythrosin; and thermally activatable molecules selected from thegroup consisting of 4,4' azobis(4-cyanopentanoic) acid, and2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride, and benzoylperoxide; and the pendent polymerizable groups are selected from thegroup consisting of vinyl groups, acrylate groups, methacrylate groups,ethacrylate groups, 2-phenyl acrylate groups, acrylamide groups,methacrylamide groups, itaconate groups, and styrene groups, wherein thesystem comprises polymers bearing both initiator groups andpolymerizable groups in combination with polymers providing only pendentpolymerizable groups.
 39. A crosslinkable macromer system comprising oneor more polymers providing pendent polymerizable and pendent initiatorgroups wherein the system is adapted to be polymerized in order to forma matrix suitable for in vivo application, and wherein the initiatorgroups are independently selected from the group consisting of long waveultraviolet activatable molecules selected from the group consisting ofbenzophenone, thioxanthones, and benzoin ethers; visible lightactivatable molecules selected from the group consisting of ethyl eosin,eosin Y, rose bengal, camphorquinone and erythrosin; and thermallyactivatable molecules selected from the group consisting of 4,4'azobis(4-cyanopentanoic) acid, and 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and benzoyl peroxide; and the pendentpolymerizable groups are selected from the group consisting of vinylgroups, acrylate groups, methacrylate groups, ethacrylate groups,2-phenyl acrylate groups, acrylamide groups, methacrylamide groups,itaconate groups, and styrene groups, wherein the groups are pendentupon biodegradable polymers adapted to form hydrogel matrices.
 40. Amacromer system according to claim 39 wherein the polymer is anaturally-occurring polymer selected from the group consisting ofpolysaccharides and polyamino acids.
 41. A macromer system according toclaim 40 wherein the polysaccharides are selected from the groupconsisting of hyaluronic acid, starch, dextran, heparin, and chitosan,and the polyamino acids are selected from the group consisting ofgelatin, collagen, fibronectin, laminin, albumin and active peptidedomains thereof.
 42. A crosslinkable macromer system comprising one ormore polymers providing pendent polymerizable and pendent initiatorgroups wherein the system is adapted to be polymerized in order to forma matrix suitable for in vivo application, and wherein the initiatorgroups are independently selected from the group consisting of long waveultraviolet activatable molecules selected from the group consisting ofbenzophenone, thioxanthones, and benzoin ethers; visible lightactivatable molecules selected from the group consisting of ethyl eosin,eosin Y, rose bengal, camphorquinone and erythrosin; and thermallyactivatable molecules selected from the group consisting of 4,4'azobis(4-cyanopentanoic) acid, and 2,2-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and benzoyl peroxide; and the pendentpolymerizable groups are selected from the group consisting of vinylgroups, acrylate groups, methacrylate groups, ethacrylate groups,2-phenyl acrylate groups, acrylamide groups, methacrylamide groups,itaconate groups, and styrene groups, wherein the polymer comprises asynthetic polymer prepared via condensation polymerization of one ormore monomers.
 43. A macromer system according to claim 42 wherein thepolymers are hydrophobic, biodegradable polymers selected from the groupconsisting of polylactides, polyglycolides, polycaprolactones, andcopolymers of any combination of lactides, glycolides and caprolactones,and further consisting of polyanhydrides and polyortho esters.
 44. Acrosslinkable macromer system comprising one or more polymers providingpendent polymerizable and pendent initiator groups wherein the system isadapted to be polymerized in order to form a matrix suitable for in vivoapplication, and wherein the initiator groups are independently selectedfrom the group consisting of long wave ultraviolet activatable moleculesselected from the group consisting of benzophenone, thioxanthones, andbenzoin ethers; visible light activatable molecules selected from thegroup consisting of ethyl eosin, eosin Y, rose bengal, camphorquinoneand erythrosin; and thermally activatable molecules selected from thegroup consisting of 4,4' azobis(4-cyanopentanoic) acid, and2,2-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride, and benzoylperoxide; and the pendent polymerizable groups are selected from thegroup consisting of vinyl groups, acrylate groups, methacrylate groups,ethacrylate groups, 2-phenyl acrylate groups, acrylamide groups,methacrylamide groups, itaconate groups, and styrene groups, wherein thepolymers further comprise pendent reactive groups or affinity groups,and wherein the reactive groups or affinity groups are adapted to bindto target groups on a surface of interest.